Mononucleotide repeats microsatellite markers for detecting microsatellite instability

ABSTRACT

A method for evaluating microsatellite instability associated with a tumour, which entails the steps of amplifying microsatellite loci in a biological sample containing genomic DNA from the tumour and determining sizes of DNA amplification products, wherein at least one microsatellite locus selected from the group consisting of NR 21, NR 22, NR 24 and NR 27, is amplified.

The present application is based on, and claims priority from, EuropeanApplication No. 02290483.3, filed Feb. 28, 2002, the disclosure of whichis hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention concerns new microsatellite markers and their use for thedetection of the microsatellite instability (MSI) associated with sometumours.

BACKGROUND OF THE INVENTION

Microsatellites are short DNA motifs (1-10 base pairs), which occur astandem repeats at numerous loci throughout the genome.

The microsatellite instability (MSI) phenotype is defined as thepresence in tumour DNA of alternative sized microsatellites that are notseen in the corresponding germline DNA (AALTONEN et al., Science,260(5109), 812-816, 1993; IONOV et al., Nature, 363(6429), 558-561,1993; THIBODEAU et al., Science, 260(5109), 816-819, 1993; IACOPETTA etal., Hum. Mutat., 12(5), 355-360, 1998).

The MSI phenotype is a characteristic of the hereditary non-polyposiscolorectal cancer (HNPCC) syndrome, wherein it can be detected in morethan 90% of all HNPCC tumours (LIU et al., Nature Med., 2, 169-174,1996); it also occurs in approximately 15% of sporadic colon and gastrictumours. It has also been detected in other tumours, such as pancreaticcarcinomas (HAN et al., Cancer Res., 53, 5087-5089, 1993), prostatecarcinomas (GAO et al., Oncogene, 9, 2999-3003, 1994), carcinomas of theendometrium (RISINGER et al., Cancer Res., 53, 5100-5103, 1993;PELTOMAKI et al., Cancer Res., 53, 5853-5855, 1993).

MSI reflects an underlying mismatch repair (MMR) defect that fails torecognize errors introduced during the replication of microsatellitesequences. In the familial cancer syndrome HNPCC, the MSI phenotype iscaused by germline mutations in the mismatch repair (MMR) genes hMSH2,HMLH1 and less frequently in hPMS1, hPMS2 and hMSH6 (KINZLER et al.,Cell, 87, 159-170, 1996). In sporadic cancers it is often caused bymethylation of the hMLH1 promoter leading to the transcriptionalsilencing of this gene (HERMAN et al., Proc. Natl. Acad. Sci. USA,95(12), 6870-6875, 1998).

MSI colonic and gastric tumours have distinctive molecular andclinicopathological profiles and are often associated with favourableprognosis (LOTHE et al., Cancer Res., 53, 5849-5852, 1993; KIM et al.,Am. J. Pathol., 145, 148-156, 1994; OLIVEIRA et al., Am. J. Pathol.,153, 1211-1219, 1998). There is also evidence to suggest that colorectalcancer patients with MSI tumours show good survival benefit from5FU-based chemotherapy (ELSALEH et al., The Lancet, 355, 1745-1750,2000; LIANG et al., Int. J. Cancer, 101, 519-525, 2002) and thereforeMSI might be a useful molecular predictive marker for response to thistype of adjuvant therapy. Routine analysis of MSI status also hasclinical application for assisting in the diagnosis of suspected HNPCCcases (AALTONEN et al., N. Eng. J. Med., 338, 1481-1487, 1998). Indeed,tumours from HNPCC patients lack phenotypic features that readilydistinguish them from sporadic tumours and hence the diagnosis of thisdisease was historically based on family history of cancer using forexample the Amsterdam criteria (VASEN et al., Dis. Colon Rectum, 34,424-425, 1991; VASEN et al., Gastroenterology, 115, 1453-1456, 1999).Such criteria are too restrictive however and identify only a fractionof HNPCC families so that the true incidence of this disease is notknown and estimates vary from 0.5 to 13%. Given that familial carriersof MMR defects have a greater than 80% risk of developing cancer, it isimportant to devise efficient and cost-effective ways to detect thiscondition. For this purpose, molecular-based laboratory approaches arenow being developed that may help in establishing HNPCC diagnosis. Twomethods are generally proposed: microsatellite genotyping andimmunohistochemistry of the main mismatch repair proteins. The use ofone or the other, or both of these methods is still a matter of debate,based on their relative efficiency, specificity and cost (LINDOR et al.,J. Clin. Oncol., 20, 1043-1048, 2002; WAHLBERG et al., Cancer Res., 62,3485-3492, 2002; TERDIMAN et al., Gastroenterology, 120, 21-30, 2001;LOUKOLA et al., Cancer Res., 61, 4545-4549, 2001). It appears so farthat microsatellite genotyping has a higher sensitivity than IHC, but ismore expensive and more difficult to set up in routine laboratories. Itis thus important to develop simple and accurate methods to determineMSI tumours for predisposition and prognostic diagnosis informations.

Numerous different microsatellites have been studied by investigatorswith the aim of identifying MSI tumours.

Depending on the type and number of microsatellites analysed, widelyvariable results for the frequency of MSI in different tumour types havebeen published (PERUCHO, Cancer Res., 59(1), 249-256, 1999).

The use of a BAT-25 and BAT-26 marker combination has been proposed forthe detection of MSI (ZHOU et al., Genes, Chromosomes & Cancer, 21(2),101-107, 1998; HOANG et al., Cancer Res., 57(2), 300-303, 1997).

The BAT-25 and BAT-26 are mononucleotide repeats respectively located inintron 16 of c-kit and intron 5 of hMSH2. These two repeats arequasimonomorphic in Caucasian populations (HOANG et al., Cancer Res.,57(2), 300-303, 1997; ZHOU et al., Oncogene, 15(14), 1713-1718, 1997).This property allows ready classification of the large allelic sizevariations seen in MSI tumour DNA as being due to somatic alteration. Inthe large majority of tumours, analysis of BAT-25 and BAT-26 issufficient to establish their MSI status without reference to thegermline DNA (ZHOU et al., Genes, Chromosomes & Cancer, 21(2), 101-107,1998).

However, alternative sized BAT-25 and BAT-26 alleles have beenidentified in 18.4 and 12.6%, respectively, of African Americans (PYATTet al., Am. J. Pathol., 155(2), 349-353, 1999; SAMOWITZ et al., Am. J.Pathol., 154(6), 1637-1641, 1999). Thus, analysis of additional repeatsmay be needed in order to avoid the occasional false positive resultarising from these germline polymorphisms.

Accordingly it has been proposed to complete the analysis of thesemononucleotide repeats by an additional analysis of dinucleotide repeatsin both the tumour and germline DNA.

For instance, U.S. Pat. No. 6,150,100 proposes the use of 2mononucleotide repeats selected from BAT25, BAT26 and BAT40, associatedwith 2 or 3 dinucleotide repeats selected from APC, Mfd15, D2S123, andD18S69, and optionally with the pentanucleotide repeat TP53Alu.Preferred combinations of microsatellite markers disclosed in U.S. Pat.No. 6,150,100 are BAT25, BAT26, APC, Mfd15 and D2S123 or BAT26, BAT40,APC, Mfd15 and D2S123.

In 1997 an international consensus meeting on the detection of MSIrecommended a panel of five markers for the uniform analysis of MSIstatus (BOLAND et al., Cancer Res., 58(22), 5248-5257, 1998). Thisincluded two mononucleotide (BAT-25 and BAT-26) and three dinucleotide(D5S346, D2S123 and D17S250) repeats. Tumours with instability at two ormore of these markers were defined as being MSI-H. Tumours withinstability at one marker, and without instability were defined as MSI-Land MSS respectively. MSI-H cancers have distinct clinicopathologicalfeatures from MSI-L and MSS tumours.

Some of the characteristics of dinucleotide repeats make their use asmarkers of the MSI status somewhat problematical. The dinucleotiderepeats in the above panels generally show instability in only 60-80% ofMSI-H tumours (SUTTER et al., Mol. Cell Probes, 13(2), 157-165, 1999).There is some evidence to suggest that loss of MMR and subsequentalteration of mononucleotide repeats occurs earlier in the MSI-H tumourprogression pathway than the mutation of dinucleotide repeats (PERUCHOet al., Cold Spring Harb. Symp. Quant. Biol., 59, 339-348, 1994).Furthermore, some MSI cell lines with MMR deficiency caused by hMSH6mutation do not show alteration in dinucleotide repeats (AKIYAMA et al.,Cancer Res., 57(18), 3920-3923, 1997). Therefore the underlying MMRdeficiency affecting mononucleotide and dinucleotide repeats may bedifferent and the analysis of both may lead to misinterpretation of theMSI status of some tumours. In many instances the analysis ofdinucleotide repeats adds no further information to the results obtainedby analysis of mononucleotide repeats (DIETMAIER et al., Cancer Res.,57(21), 4749-4756, 1997; LOUKOLA et al., Cancer Res., 61(11), 4545-4549,2001).

In addition, unlike mononucleotide repeats such as BAT-25 and BAT-26,dinucleotide repeats are highly polymorphic. Therefore, their use forthe identification of MSI in tumour DNA always requires the analysis ofcorresponding germline DNA.

This makes the MSI screening process considerably more time-consumingand expensive, as well as introducing potential errors due to mixing ofgermline and tumour DNA samples. Moreover, the interpretation of sizealterations in these dinucleotide repeats is difficult and can lead tomisclassification (PERUCHO, Cancer Res., 59(1), 249-256, 1999). Finally,in many situations germline DNA from cancer patients is not readilyavailable.

For all these reasons, the methods using BAT-26 and BAT-25 either alone,or in combination with dinucleotide repeats are not completelysatisfactory for the accurate determination of MSI status in humantumours and there is an urgent need for improvement.

Thus, multiple, quasimonomorphic mononucleotide repeats are needed forthe accurate diagnosis of MSI tumours.

SUMMARY OF THE INVENTION

The inventors have now identified new mononucleotide repeats that areconserved in germline DNA from Caucasian and African subjects and that,similar to BAT-25 and BAT-26, are highly sensitive to somatic deletionin MSI-H tumours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows typical allelic profiles of (a) BAT 25, BAT 26, NR 21, NR22 and NR 24 in DNA from the germline or from MSS tumours, (b) MSI-Hprimary tumour showing both deleted and normal sized alleles, and (c)MSI-H cell line showing homozygous deletion.

FIG. 1 a shows an example of the fluorescent peaks observed for eachmarker, in this case representing the most common allele found ingermline DNA.

FIG. 1 b shows the size of PCR products and corresponding fluorescentlabels which were chosen so as to allow simultaneous analysis ofnormal-sized alleles with the smaller-sized alleles that are typicallyseen in MSI-H tumours.

FIG. 1 c shows that in addition to the smaller alleles, most MSI-Hprimary tumours also showed normal-sized alleles presumably originatingfrom contaminating non-cancer cells.

FIG. 2 shows that none of the germline DNA samples tested werepolymorphic in more than 2/5 markers.

FIG. 3 shows that a total of 104 colon and gastric tumours and celllines which were previously identified as MSI-H showed amplificationdata for all five markers. Tumours showed deletions in either all (88tumours) or 4/5 (9 tumours) mononucleotide repeats.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Three of these new microsatellite markers are poly(T) repeatshereinafter referred as NR21, NR22, and NR24.

The NR21 marker is a 21T repeat identified in the 5′ untranslated regionof the SLC7A8 gene (cDNA sequence GenBank XM_(—)033393).

The NR22 marker is a 22T repeat identified in the 3′ untranslated regionof the putative trans-membrane precursor protein B5 gene (cDNA sequenceGenBank L38961).

The NR24 marker is a 24T repeat identified in the 3′ untranslated regionof the zinc finger-2 gene (cDNA sequence GenBank X60152).

A fourth microsatellite marker, hereinafter referred as NR27, is a 27Arepeat identified in the 5′ untranslated region of the inhibitor ofapoptosis protein-1 gene (cDNA sequence GenBank AF070674).

The NR21, NR22, NR24 and NR27 markers are useful for the evaluation ofmicrosatellite instability in the diagnosis of tumours.

The invention thus provides a method for evaluating the microsatelliteinstability associated with a tumour, by amplification of microsatelliteloci in a biological sample comprising genomic DNA from said tumour anddetermination of the sizes of the DNA amplification products,characterized in that said method comprises the amplification of atleast one microsatellite locus selected among NR21, NR22, NR24 and NR27.

According to a preferred embodiment of the invention, said methodcomprises the amplification of the two microsatellite loci NR21 andNR24, and the amplification of a third microsatellite locus selectedamong NR22 and NR27.

Advantageously, said method further comprises the amplification of atleast one microsatellite locus different from NR21, NR22, NR24 and NR27.Preferably, said microsatellite locus is a mononucleotide repeat locus.More preferably this mononucleotide repeat locus is selected amongBAT-25 and BAT-26.

According to a particular embodiment, the method of the inventioncomprises the amplification of the five microsatellite loci BAT-25,BAT-26, NR21, NR22, and NR24.

According to another particular embodiment, the method of the inventioncomprises the amplification of the five microsatellite loci BAT-25,BAT-26, NR21, NR27 and NR24.

Microsatellite instability at each of these loci is evaluated bycomparison of the size of the amplification product obtained fromtumoral DNA with the size of the amplification product obtained fromnormal (i.e. non-tumoral) DNA with the same set of primers.

This comparison can be performed in the conventional way, by obtainingan amplification product from normal DNA from the same subject with thesame set of primers, and using it as a reference.

However, the present invention makes it possible, in most of cases, toavoid the need to amplify normal DNA from the same subject. Instead, thecomparison can be made by reference to the average size of amplificationproducts obtained from normal DNAs of a pool of subjects with the sameset of primers. In these cases, microsatellite instability is assumed inthe case of locus BAT-26 if the size of the amplification productobtained from tumoral DNA is shorter of more than 3 bp than the averagesize of the amplification product obtained from normal DNA using thesame set of primers, and in the case of loci BAT-25, NR21, NR22, NR24and NR27, if the size of the amplification product obtained from tumoralDNA is shorter of more than 2 bp than the average size of theamplification product obtained from normal DNA using the same set ofprimers.

Tumoral genomic DNA can be obtained from different sources includingprincipally biopsies or tumoral tissues, or body fluids or secretionscontaining disseminated tumour cells, paraffin embedded tissue.

The invention also provides reagents for carrying out the method of theinvention.

In particular, the invention provides pairs of primers suitable for theamplification of a microsatellite locus selected among NR21, NR22, NR24and NR27.

Suitable primers can be derived from the genomic sequences surroundingsaid microsatellite loci.

For instance:

primers allowing the amplification of NR21 can be derived from thegenomic sequence GenBank AL117258, and preferably from the portionthereof represented by SEQ ID NO: 1;

primers allowing the amplification of NR22 can be derived from thegenomic sequence GenBank AP001132, and preferably from the portionthereof represented by SEQ ID NO: 2;

primers allowing the amplification of NR24 can be derived from thegenomic sequence GenBank AC092835, and preferably from the portionthereof represented by SEQ ID NO: 3;

primers allowing the amplification of NR27 can be derived from thegenomic sequence GenBank AP001167, and preferably from the portionthereof represented by SEQ ID NO: 16.

By way of example:

a pair of primers suitable for the amplification or NR21 consists of thefollowing oligonucleotides:

TAAATGTATGTCTCCCCTGG (SEQ ID NO: 4) ATTCCTACTCCGCATTCACA (SEQ ID NO: 5)

a pair of primers suitable for the amplification or NR22 consists of thefollowing oligonucleotides:

GAGGCTTGTCAAGGACATAA (SEQ ID NO: 6) AATTCGGATGCCATCCAGTT (SEQ ID NO: 7)

a pair of primers suitable for the amplification or NR24 consists of thefollowing oligonucleotides:

CCATTGCTGAATTTTACCTC (SEQ ID NO: 8) ATTGTGCCATTGCATTCCAA. (SEQ ID NO: 9)

The above primers give when used on normal DNA, amplification productsof 104, 143, and 134 bp for NR21, NR22 and NR24 respectively.

They can advantageously be labelled with fluorescent dyes and used inmultiplex PCR assays. Preferably, different fluorescent dyes will beused for primers that give amplification products of similar size (i.ehaving sizes differing of less than 15-20 pb). This allows to avoiduncertainties that might result from overlapping of PCR products due tothe average deletion of 5-12 bp for these markers in MSI tumors.

If one prefers not to use different fluorescent dyes, primers can bedesigned in order to give amplification products of clearly distinctsize (i.e having sizes differing of at least 15 pb and preferably of atleast 20 pb between different markers). This allows a clear separationbetween markers on a size basis by standard electrophoresis techniques,even when deleted due to microsatellite instability in tumor DNA.

Primers giving amplification products of clearly distinct size for NR21,NR24, and NR27 are by way of example:

a pair of primers suitable for the amplification or NR21 consists of thefollowing oligonucleotides:

GAGTCGCTGGCACAGTTCTA; (SEQ ID NO: 17) CTGGTCACTCGCGTTTACAA; (SEQ ID NO:18)

a pair of primers suitable for the amplification or NR24 consists of thefollowing oligonucleotides:

GCTGAATTTTACCTCCTGAC; (SEQ ID NO: 19) ATTGTGCCATTGCATTCCAA; (SEQ ID NO:9)

a pair of primers suitable for the amplification or NR27 consists of thefollowing oligonucleotides:

AACCATGCTTGCAAACCACT; (SEQ ID NO: 20) CGATAATACTAGCAATGACC. (SEQ ID NO:21)

When used on normal DNA, the above primers give amplification productsof 131, 109 and 87 bp for NR24, NR21 and NR27 respectively.

The invention also provides a kit for the analysis of microsatelliteinstability, characterized in that it comprises at least two pairs ofprimers suitable for the amplification of at least two microsatelliteloci selected among NR21, NR22, NR24, and NR27.

Advantageously said kit comprises at least:

one pair of primers suitable for the amplification of NR21;

one pair of primers suitable for the amplification of NR24;

one pair of primers selected among a pair of primers suitable for theamplification of NR22 and a pair of primers suitable for theamplification of NR27.

According to a preferred embodiment said kit further comprises at leastone pair of primers suitable for the amplification of at least onemicrosatellite locus different from NR21, NR22, NR24 and NR27.Preferably, said microsatellite locus is a mononucleotide repeat locus.More preferably this mononucleotide repeat locus is selected amongBAT-25 and BAT-26.

Primers allowing the amplification of BAT-26 can be derived from thegenomic sequence GenBank AC0799775, and preferably from the portionthereof represented by SEQ ID NO: 10

Primers allowing the amplification of BAT-25 can be derived from thegenomic sequence GenBank AC092545, and preferably from the portionthereof represented by SEQ ID NO: 11

By way of example, a kit of the invention can comprise:

a pair of primers suitable for the amplification of BAT-25, consistingof the following oligonucleotides:

TCGCCTCCAAGAATGTAAGT (SEQ ID NO: 12) TCTGCATTTTAACTATGGCTC; (SEQ ID NO:13)

a pair of primers suitable for the amplification of BAT-26, consistingof the following oligonucleotides:

TGACTACTTTTGACTTCAGCC (SEQ ID NO: 14) AACCATTCAACATTTTTAACCC; (SEQ IDNO: 15)

When used on normal DNA, the above primers amplify a fragment of 121 bpfor BAT-26, and 124 bp for BAT-25.

They can be used in particular in a multiplex PCR assay using differentfluorescent dyes, for instance in combination with the NR21 primers SEQID NO: 4 and 5, the NR22 primers SEQ ID NO: 6 and 7, and the NR24primers SEQ ID NO: 8 and 9.

If one prefers to obtain amplification products of clearly distinct sizefor BAT-25 and BAT-26, one can use for instance:

a pair of primers suitable for the amplification or BAT-25 consisting ofthe following oligonucleotides:

TACCAGGTGGCAAAGGGCA; (SEQ ID NO: 22) TCTGCATTTTAACTATGGCTC; (SEQ ID NO:13)

a pair of primers suitable for the amplification or BAT-26, consistingof the following oligonucleotides:

CTGCGGTAATCAAGTTTTTAG; (SEQ ID NO: 23) AACCATTCAACATTTTTAACCC. (SEQ IDNO: 15)

When used on normal DNA, the above primers give respectivelyamplification products of 153 and 183 bp for BAT-25 and BAT-26.

They can advantageously be used in combination with NR21 primers (SEQ IDNO: 17 and 18) and/or NR24 primers (SEQ ID NO: 19 and 9) and/or NR27primers (SEQ ID NO: 20 and 21), allowing a clear separation of the fivemarkers on a size basis.

Optionally, the kits of the invention can further comprise appropriatereagents and materials useful to carry out DNA amplification.

The method, reagents and kits of the invention can be used in the sameapplications as the prior art methods of evaluation of microsatelliteinstability. This includes mainly the diagnosis of the MSI phenotype oftumours, in particular tumours of the gastrointestinal tract, and morespecifically colorectal or gastric tumours, or tumours of theendometrium. Tumours with instability at three or more of the BAT-25,BAT-26, NR21, NR22 (or NR27), or NR24 loci are defined as being MSI-H.

The method of the invention has the advantage over the prior art methodsof allowing to establish the MSI status without ambiguity in particularin the case of tumours of the gastrointestinal tract, without needing asimultaneous analysis of corresponding germline DNA from each patient.

We propose that concurrent use of these mononucleotide markers in asingle pentaplex PCR system allows accurate evaluation of tumour MSIstatus with 100% sensitivity, 100% specificity. This assay is simpler touse than those involving dinucleotide markers, and is more specific thanusing BAT-25 and BAT-26 alone. This test could be routinely used in thehospital to provide information on prognosis, as a possible predictor ofresponse to adjuvant therapies, and for the detection of new HNPCCfamily members.

The invention will be further illustrated by the additional descriptionwhich follows, which refers to an example of use of the mononucleotidemarkers of the invention in multiplex PCR analysis. It should beunderstood however that this example is given only by way ofillustration of the invention and does not constitute in any way alimitation thereof.

Example Material and Methods Mononucleotide Repeats and MultiplexPolymerase Chain Reaction (PCR)

Three new poly(T) repeats and one new poly(A) repeat were identifiedrespectively in the 3′ or 5′ untranslated regions of the SLC7A8 (NR21,21T), trans-membrane precursor protein B5 (NR22, 22T), zinc finger-2(NR24, 24T), and inhibitor of apoptosis protein-1 (NR27, 27A) genes.Details including primer sequences for these repeats and BAT 25 and BAT26 are shown in Table I below.

TABLE I GenBank SEQ PCR accession Location of the Fluorescent ID productName Gene (cDNA) repeat marker colour Primers NO (bp)* BAT 26 hMSH2U04045 26(A)intron 5 FAM^(a) blue TGACTACTTTTGACTTCAGCC 14 121AACCATTCAACATTTTTAACCC 15 CTGCGGTAATCAAGTTTTTAG 23 183AACCATTCAACATTTTTAACCC 15 BAT 25 c-kit X06182 25(T)intron 16 NED^(s)yellow TCGCCTCCAAGAATGTAAGT 12 124 TCTGCATTTTAACTATGGCTC 13TACCAGGTGGCAAAGGGCA 22 153 TCTGCATTTTAACTATGGCTC 13 NR21 SLC7A8XM_033393 21(T)5′UTR HEX^(a) green TAAATGTATGTCTCCCCTGG 4 104ATTCCTACTCCGCATTCACA 5 GAGTCGCTGGCACAGTTCTA 17 109 CTGGTCACTCGCGTTTACAA18 NR22 Putative trans- L38961 22(T)3′UTR FAM^(a) blueGAGGCTTGTCAAGGACATAA 6 143 membrane pre- AATTCGGATGCCATCCAGTT 7 cursorprotein B5 NR24 ZINC FINGER 2 X60152 24(T)3′UTR HEX^(a) greenCCATTGCTGAATTTTACCTC 8 134 (ZNF-2) ATTGTGCCATTGCATTCCAA 9GCTGAATTTTACCTCCTGAC 19 131 ATTGTGCCATTGCATTCCAA 9 NR27 Inhibitor ofAF070674 27(A)5′UTR AACCATGCTTGCAAACCACT 20 87 apoptosisCGATAATACTAGCAATGACC 21 protein-1 ^(s)sense primer ^(a)anti-sense primer*theorical size deduced from GenBank sequence

Primers were designed to allow different PCR product sizes to beresolved on 5% denaturing gels run in an ABI PRISM 377 automated DNAsequencer. GENESCAN software (GENOTYPER 2.1) was used to calculate thesize, height and area of each fluorescent PCR product.

The following primers were used in the experimentations described below:

BAT 26 primers: SEQ ID NO: 14 and SEQ ID NO: 15;

BAT 25 primers: SEQ ID NO: 12 and SEQ ID NO: 13;

NR21 primers: SEQ ID NO: 4 and SEQ ID NO: 5;

NR22 primers: SEQ ID NO: 6 and SEQ ID NO: 7;

NR24 primers: SEQ ID NO: 8 and SEQ ID NO: 9.

One primer in each pair was labelled with one of the fluorescent markersFAM, HEX or NED (PE APPLIED BIOSYSTEMS). The five mononucleotide repeatswere amplified in one multiplex PCR containing 20 μM of each primer, 200μM dNTP, 1.5 MM MgCl₂ and 0.75 units of Taq DNA polymerase. The PCR wasperformed using the following conditions: denaturation at 94° C. for 5min, 35 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C.for 30 sec and extension at 72° C. for 30 sec, followed by an extensionstep for 72° C. for 7 min.

DNA Samples

Germline DNA was obtained from 128 Caucasian individuals at the Centred'Etudes du Polymorphisme Humain (CEPH) in Paris and from 56 individualsof African descent.

A total of 124 colon, 50 gastric tumours, 20 endometrial tumours and 16colon cell lines that had previously been tested for MSI using severaldinucleotide markers and BAT-25 and BAT-26 mononucleotide markers (147cases) or BAT26 and BAT25 alone (63 cases) (HOANG et al. Cancer Res.,57(2), 300-303, 1997 ; SERUCA et al., Int. J. Cancer, 64, 32-36, 1995;TIBELETTI et al., Gynecol. Oncol., 73(2), 247-252, 1999). Of these atotal of 81 primary colon tumours, 42 primary gastric cancers, 20primary endometrial tumours and 5 colon tumour cell lines wereconsidered to be MSI-H based on deletions in the above repeats.

Results Fluorescent Multiplex PCR

The five mononucleotide markers BAT-25, BAT-26, NR21, NR22 and NR24 wereco-amplified in a single multiplex PCR mix using the PCR conditionsdescribed above, and analysed for size in an automated DNA sequencer. Inthese conditions, no non-specific bands within the 100-142 bp size rangewere observed, thus allowing accurate identification of the fivemarkers.

FIG. 1 shows typical allelic profiles of (a), BAT25, BAT26, NR21, NR22and NR24 in DNA from the germline or from MSS tumours, (b) MSI-H primarytumour showing both deleted and normal sized alleles, and (c) MSI-H cellline showing homozygous deletions.

FIG. 1 a shows an example of the fluorescent peaks observed for eachmarker, in this case representing the most common allele size found ingermline DNA.

The size of PCR products and the corresponding fluorescent labels werechosen so as to allow simultaneous analysis of normal sized alleles withthe smaller sized alleles containing deletions that are typically seenin MSI-H tumours (FIG. 1 b). In addition to the smaller alleles mostMSI-H primary tumours also showed normal sized alleles that presumablyoriginate from contaminating non-cancer cells. These were absent in thehomozygous mutant MSI-H cell line shown in FIG. 1 c.

The most common allelic sizes for BAT-25, BAT-26, NR21, NR22 and NR24were 124, 120, 103, 142 and 132 bp respectively, although for eachrepeat, a slight variation in the position of the peaks representing thesize of the PCR product was observed (FIG. 2).

Legend of FIG. 2:

=BAT-25

▪=BAT-26

=NR21

=NR22

=NR24

In order to account for these variations, for BAT-25, NR21, NR22 andNR24 alleles of ≧3 pb and for BAT-26 allelic sizes of ≧4 pb wereconsidered to be polymorphisms or somatic alterations.

Polymorphisms in Germline DNA

As shown in Table IIa below, each marker was at least 95% monomorphic in128 germline DNA samples from unrelated Caucasians (CEPH samples).

TABLE IIa BAT-26 BAT-25 NR21 NR22 NR24 germline germline DNA 128/128126/128 122/128 128/128 128/128 Caucasian (100%) (98.4%) (95.3%) (100%)(100%) germline germline DNA 51/56 44/56 54/56 56/56 52/56 African(91.1%)  (78.6%) (96.1%) (100%) (92.9%) 

Furthermore, 121 (94.5%) of this population was monomorphic in all fiverepeats and the remaining 7 individuals (5.5%) were monomorphic in 4/5markers. No CEPH DNA sample contained a polymorphism in more than 1 ofthe 5 repeats. Polymorphisms were more common in African germline DNA,with BAT 25 having the highest level of polymorphism at 21.4%. Althoughinterestingly, of 56 African germline DNA samples tested, 37 (66.1%)were monomorphic in all five repeats, 15 (26.8%) showed a polymorphismin 1/5 markers and 4 (7.1%) in 2/5 markers. None of the germline DNAsamples were polymorphic in >2/5 markers. These data are also shown inFIG. 3.

Identification of MSI-H Tumours

Using the above criteria the average deletion observed for eachmononucleotide repeat was calculated in MSI-H gastric and colon tumours.The sensitivity, specificity and the average deletions of the fivemarkers in different types of DNA are shown in Table IIb below.

TABLE II BAT-26 BAT-25 NR21 NR22 NR24 Colon average deletion   11.9 7.36.8 5.1 5.6 MSI-H (sensitivity) 77/77 72/72 78/78 77/79 72/75 (100%)(100%) (100%) (97.5%)   (96%) MSS (specificity) 42/43 44/44 43/44 45/4543/44 (97.7%)  (100%) (97.7%)   (100%) (97.7%) Colon Cell Line averagedeletion 11 7.8 7.4 4.4 7.6 MSI-H (sensitivity) 4/4 5/5 5/5 5/5 5/5(100%) (100%) (100%)  (100%)  (100%) MSS (specificity) 11/11 11/11 11/1111/11 10/10 (100%) (100%) (100%)  (100%)  (100%) Gastric averagedeletion 12 7.2 6.8 4.9 5   MSI-H (sensitivity) 39/39 39/39 37/39 38/3926/30 (100%) (100%) (94.9%)  (97.4%) (86.7%) MSS (specificity)  8/1110/11 10/10 10/11 11/11 (72.7%)  (90.9%)  (100%) (90.9%)  (100%)Endometrial average deletion   5.9 4.6 3.9 2.9 2.4 MSI-H (sensitivity)15/17 18/19 17/19 14/19 10/16 (88.2%)  (94.7%)  (89.5%)  (73.7%) (62.5%)

For BAT-26, the average deletion was almost 12 bp, or approximatelytwice the average length of deletion seen with the other markers. Eachmononucleotide repeat was deleted in MSI-H tumours with asensitivity >86%. Allelic shifts due to polymorphisms or somaticmutation were infrequent in non-MSI tumours, resulting in a high degreeof specificity for the detection of MSI by each of these markers, withthe exception of BAT-26 whose specificity was lowered due to theprevious misclassification of 6 tumours as discussed below.

A total of 104 colon and gastric tumours and cell lines which werepreviously identified as MSI-H showed amplification data for all fivemarkers. Tumours showed deletions in either all (88 tumours) or 4/5 (9tumours) mononucleotide repeats (FIG. 3). Only one sample showeddeletions in 3/5 markers. In 5 cases, previously defined as MSI-H, sizealterations were found in only Bat-26 or Bat-25; these samples wereconsidered as misclassified due to ethnic polymorphisms (4 cases) orborderline shortening (1 case). Finally, an additional tumour sample waspreviously classified as MSI-H using dinucleotide repeats but not BAT-26(HOANG et al., Cancer Res., 59(1), 300-303, 1997). This sample wasmonomorphic for all five mononucleotide repeats used in this study,suggesting that it was misclassified with dinucleotide repeats, possiblydue to the fact that germline DNA did not match with tumour DNA. InTable II and FIG. 3, these 6 samples were considered as MSS.

Of 55 colon and gastric tumours and cell lines previously classified asMSS and containing data for all 5 repeats, 53 (96.4%) were monomorphicat all 5 markers. One tumour was monomorphic at 4 markers (2%) and 1tumour was monomorphic at 3/5 (2%) repeats. None of the 55 MSS tumoursshowed allelic shifts in 3 or more repeats (FIG. 3), still complyingwith the MSI identification criteria of 2/5 repeat polymorphismsdescribed earlier.

These results are illustrated by FIG. 3, which shows the percentage ofsamples with allelic size shifts in the five mononucleotide repeats inCaucasian germline DNA (□), African germline DNA (▪), MSS tumours (

), MSI-H colorectal tumours (

) and MSI-H gastric tumours (

).

The three mononucleotide repeats NR21, NR22, and NR24 arequasimonomorphic in germline DNA and, similar to BAT-25 and BAT-26, arehighly sensitive to somatic deletion in MSI-H tumours. Distinction ofMSI-H from MSS tumours is unambiguous when these three new markers areused in conjunction with BAT-25 and BAT-26. Multiplex PCR of these 5markers has the additional advantage of avoiding the need forsimultaneous analysis of corresponding germline DNA from each patient.

Although the quasimonomorphic nature of the three new mononucleotiderepeats remains to be fully established in different populations, noneof 128 Caucasian and 56 African germline DNA cases had polymorphisms inmore than 2 of the repeats. Since all 98 true MSI-H tumours examinedhere, with successful amplification of the 5 markers, showed deletionsin at least 3 markers, the probability of misinterpretation of an MSIresult because of polymorphisms in 3 or more of the 5 markers isstatistically insignificant. Therefore when the results from all fiverepeats were analysed together, the MSI status of this entire 159 tumourseries was determined unambiguously with 100% sensitivity andspecificity. Moreover, this was achieved without the need to analyzecorresponding germline DNA.

Table 3 below indicates tumours where polymorphism or borderlinedeletion on BAT-26 or BAT-25 would have misclassified the MSI status ofthe corresponding tumour. In all cases, the use of the multiplex panelallowed to unambiguously classify the tumour.

TABLE III MSI-H tumours reclassified as MSS with the pentaplex PCR MSSBAT-26 BAT-25 NR21 NR22 NR24 Polymorphisms 11 2 0 0 0 11 0 0 0 0 11 0 00 0 12 0 0 0 0 Borderline deletions 0 4 0 0 0 Unstable only in 0 0 0 0 0dinucleotide repeats

In a further 31 DNA samples from colon and gastric tumours, only fourout of the five markers were correctly amplified. This was probably dueto the quality of DNA extracted from formalin-fixed andparaffin-embedded tissues. Twenty four of these cases showed 4/4 or 3/4unstable loci and were correctly identified as MSI-H, the remaining 7cases showing 0/4 or 1/4 unstable loci were correctly identified as MSS(results not shown, but included in Table II). Therefore, incompleteamplification of the five repeats could still be used effectively toidentify the MSI status of difficult DNA.

We have previously observed that endometrial MSI-H tumours showsignificant quantitative and qualitative differences in instabilitycompared to gastro-intestinal MSI-H tumours (DUVAL et al., Cancer Res.,62, 1609-1612, 2002). In the present study, 20 endometrial tumours knownto be MSI-H were also tested with the same fluorescent pentaplex assay.These tumours showed significantly shorter average lengths of deletionsin all five mononucleotide repeats markers compared to MSI-Hgastro-intestinal tumours (Table II). Using the MSI detection criteriaestablished with this microsatellite panel, 17/20 endometrial tumourswere identified as MSI-H and 1 tumour was identified as MSS (data notshown). It was not possible to conclusively identify the MSI status ofthe remaining 2 tumours due to the very small allelic shifts observed inall five markers.

Thus, it was possible to effectively identify the MSI status of all 190colon and gastric tumours and cell lines tested in this experiment,including six samples which were previously misclassified. Because ofthe smaller size of allelic shifts found in MSI-H endometrial tumours,we recommend the continued analysis of matching germline DNA for routineMSI screening of this cancer type.

Accumulating evidence suggests that MSI status defines a subset ofcolorectal cancers with distinctive biological and clinical properties,emphasizing the importance of simple and accurate markers for detection.This set of five mononucleotide markers, determines the MSI status oftumours with higher sensitivity and specificity than BAT-25 and BAT-26alone, and is technically simpler to use than the panel recommended atthe Bethesda consensus meeting.

1) A pair of primers for amplifying a microsatellite locus selected fromthe group consisting of NR21, NR22, NR24 and NR27. 2) The pair ofprimers of claim 1 wherein said microsatellite locus is NR21, and saidprimers are obtained from SEQ ID NO:1.
 3. the pair of primers of claim1, wherein said microsatellite locus is NR22, and said primers areobtained from SEQ ID NO:2.
 4. The pair of primers of claim 1, whereinsaid microsatellite locus is NR 24, and said primers are obtained fromSEQ ID NO:3.
 5. The pair of primers of claim 1, wherein saidmicrosatellite locus is NR 27, and said pair of primers are obtainedfrom SEQ ID NO:16.
 6. The pair of primers of claim 1, which are selectedfrom the group consisting of: a) a pair of primers for amplifying NR21,comprising oligonucleotides SEQ ID NO: 4 and SEQ ID NO:5; b) a pair ofprimers for amplifying NR 21, comprising oligonucleotides SEQ ID NO: 17and SEQ ID NO:18; c) a pair of primers for amplifying NR 22, comprisingoligonucleotides SEQ ID NO:6 and SEQ ID NO:7; d) a pair of primers foramplifying NR 24, comprising oligonucleotides SEQ ID NO: 8 and SEQ IDNO: 9; e) a pair of primers for amplifying NR 24, comprisingoligonucleotides SEQ ID NO:19 and SEQ ID NO:9; and f) a pair of primersfor amplifying NR 27, comprising oligonucleotides SEQ ID NO:20 and SEQID NO:21.
 7. A kit, comprising at least two pairs of primers foramplifying at least two microsatellite loci selected from the groupconsisting of NR 21, NR 22, NR 24 and NR
 27. 8. A kit of claim 7, whichcomprises: a) one pair of primers for amplification of NR 21, b) onepair of primers for amplification of NR 24, and c) one pair of primersfor amplifying either NR 22 or NR 27 or both.
 9. The kit of any of claim7, which further comprises at least one pair of primers selected fromthe group consisting of: a) a pair of primers for amplifyingmicrosatellite locus BAT-25; and b) a pair of primers for amplifyingmicrosatellite locus BAT-26.
 10. The kit of claim 9, which comprises apair of primers for amplifying microsatellite locus BAT-25, which areoligonucleotides SEQ ID NO: 12 and SEQ ID NO:13, and oligonucleotidesSEQ ID NO:23 and SEQ ID NO:15.
 11. The kit of claim 9, which comprises apair of primers for amplifying microsatellite locus BAT-26, which areoligonucleotides SEQ ID NO:14 and SEQ ID NO:15, and oligonucleotides SEQID NO:23 and SEQ ID NO:15.
 12. The kit of claim 9, which comprises apair of primers for amplifying microsatellite locus BAT-25, and a pairof primers for amplifying microsatellite locus BAT-26.
 13. The kit ifclaim 7, which further comprises reagents for DNA amplification.
 14. Amethod for detecting amplified DNA sequences with the primers of claim1, which comprises detecting said amplified DNA sequences with adifferent fluorescent dye for each amplified DNA sequence where saidamplified sequences differ from each other by less than 20 bp in size.15. A method for detecting amplified DNA sequences with the primers ofclaim 1, which comprises detecting said amplified DNA sequences whichdiffer from each other by at least 20 bp in size.